Alkanoic acid esters of tetracycline antibiotics



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United States Patent ALKANOIC ACID ESTERS or TETRACYCLINE ANTIBIOTICS PhilipN. Gordon, Old Lyme, Comp, assignor to Chas. Pfizer & Co., Inc., New York, N. Y., a corporation of Delaware Ne Drawing. Application March 11, 1954,

Serial No. 415,691

8 Claims. (Cl. 260-490) This invention is concerned with a process for the preparation of esters of a' group of antibiotics and with the new compounds thus prepared.

The compounds oxytetracycline (also known by th registered trademark Terramycin), chlortetracycline (also knownby the trademark Aureomycin), and tetracycline (also 'known by the trademark Tetracyn), are agr'oup of substances which are highly effective antimicrobial agents. These substances have closely related structures which may be designated as follows:

In oxytetracycline R1 is OH and R2 is H; in chlortetracycline R1 is H and R2 is Cl; and in tetracycline R1 is H and R2 is H. These antibiotic compounds and their salts with acids and bases are herein designated as the tetracycline antibiotics.

'It has been shown in a co-pending patent application, Serial Number 310,559, filed by Philip N. Gordon on September 19, 1952 (now abandoned) that under certain controlled conditions oxytetracycline is selectively converted to a specific diacetate derivative; that is two of the several hydroxyls of the structure are converted to acetate ester groups. It has now been determined that in the process described and claimed in the former application, of which the present application is a continuation-in-part, the monoacetate is formed as an intermediate in the preparation of the diacetate. The intermediate Ratented Nov. 5, 1957 ified. However, since neither tetracycline nor chlortetra cycline has a hydroxyl group at the position, the reaction proceeds no further with these antibiotics under the particular conditions of the present process. The new compounds which may be prepared in accordance with the method described in this application are the 12a- 1 monoesters of tetracycline, oxytetracycline and chlormonoacetate has now been isolated, and it has. been found that the first acetyl group is introduced at the hydroxyl 12a-position of the nucleus. Upon further reaction under the controlled conditions the S-hydroxyl group is acetylated and the 5-12a-diacetate is obtained.

Not only is it possible to form the acetate esters by the above process, but by the same method utilizing higher fatty acid anhydrides, there are produced the corresponding higher esters, thus, for instance, propionic acid anhydride yields the corresponding propionates, butyric acid anhydride yields the corresponding butyrates, and valeric acid anhydride yields the corresponding valerates. In general, this method may be applied with aliphatic acid anhydrides having up to about 6 carbon atoms in their principal chain.

Not only has it been shown that in the esterification of oxytetracycline the 12a position is first esterified and then the 5 position, but this has also been shown to be true of the structurally closely related antibiotics chlortetracycline and tetracycline, that is under'thecontrolled conditions of the present process the 12a group is ester tetracycline, and the 5,12a-diesters of oxytetracycline (and the salts of these esters with acids and bases). These new compounds are useful antimicrobial agents. The diesters of oxytetracycline generally display a rather low order of in vitro microbiological activity, however, in aqueous solutions at elevated pHs the compounds hydrolyze to form the monoesters of the parent antibiotic which are highly active in vitro. There is also a certain amount of hydrolysis of the diesters when these materials are injected into animals, for instance, by the, intramuscular route. The diesters are appreciably more soluble in Water at slightly basic pHs, that is from about 7.5 to about 9, than is the parent unesterified antibiotic. The diesters also have the advantage of a higher solubility in certain solvents which may be used as vehicles for the injection of various drugs into animals, for instance the solvent propylene glycol. The ester compounds show indications of being stimulatory to plant and animal growth in addition to their antimicrobial activity.

It should be noted that the new compounds of the present invention are prepared under certain controlled conditions which permit of the formation of the esters in specific positions of the tetracycline antibiotic nucleus. Under other conditions either more highly esterified compounds or esterified compounds substituted in other positions are formed. This specificity of reaction is quite unexpected.

The following structural formula gives the general configuration of the esters which are prepared according to the present invention:

CH3 OH The monoesters of all of the compounds in the present application have at the position designated as R3 an ester group (e. g. acetoxy, propionoxy,- butyroxy, etc.). When the monoester is an ester of oxytetracycline R1 is OH and R2 is H; when it is a monoester of chlortetracycline R1 is H and R2 is Cl; 'and when it is a monoester of tetracycline R1 is H and R2 is H. In the case of the 5,12a-diesters of oxytetracycline, R3 is an ester group (e. g. acetoxy, propionoxy, butyroxy, etc.), R1 is an ester group (e. g. acetoxy, propionoxy, butyroxy, etc.) and R2 is H. The 5-monoesters and the 5,12a-diesters of the tetracycline antibiotics may be prepared by treating the parent antibiotic with the appropriate acid anhydride (e. g. acetic acid anhydride, butyric acid anhydride, propionic acid anhydride, etc.) at about room temperature, that is at from about 15 to about 35 C. It is quite important that the temperature be carefully controlled, since elevated temperatures tend to cause furher esterification of the molecule, resulting in less desirable compounds. Other side reactions may also occur. In general, an excess of the acid anhydride is preferred to achieve a reasonable rate of reaction and a homogenous reaction mixture. A concentration by weight of abouti0.5 tov 1 about 5% .of thetetracycl ne.ant b ot cin h ssts f i a reagent is generally quite suitable, however, this is not cr tical and higher or lower concentrations may be used with comparable results. The solubility of the antibiotics. vary somewhat in the various esten'fying agents. if undissolved: antibiotic is present it may be desirable to use agitation to obtain good contact between the solid and thereactio n medium. A minor proportion of certain inert organic solvents considerably assists in, controlling the rate and, extent of the esterification reaction. Of particular value are certain ethers such as dioxane whichjmay be included in the reaction mixture to facilitate the formation of the esters. A ratio of about one volume of solvent to about 4 volumes of acid anhydride has been found to; be quite satisfactory. Again, this isnot acritical condition for the? success of the reaction. The solvent assists in dissolving the solid antibiotic. and increasing the rate. of formation of the. desired product. A higher proportion of the solvent may tend to interfere with the, reaction. It should be noted that the presence of any base or a strong; acid may tend to cause the formation of undesired lay-products, or may result in the formation of esters having more than one or two ester 'as indicated above.

In theformation of the esters of the tetracyclineantibiotics by the process of the present invention, as noted above, the LZa-hydroxyl group of the nucleus is first esterified. It has been found that this product may be isolated by stopping the reaction at the appropriate stage. The timerequired' for formation of a high proportionof the monoesters may be determined withrelative ease by removing samples from a reaction mixture at regular intervals and, isolating the reaction product. Analysis of such products indicates the. extent of the esterification. It has; been found that when oxytetracycline is used as the starting material and the process is operated at a temperature of about C. ina mixture of dioxane and acetic anhydride, a high proportion of the monoacetate may be isolated by stopping the reaction at about 70 hours. It should be realized that the rate of formation is dependent upon the temperature, concentration of the tetracycline antibiotic, proportion of solvent and acetic anhydride, and other conditions. In general at last about one week is required for the formation of the diacetate compound. Within about 48 hours an appreciable amount of monoacetate is formed from the tetracycline antibiotics. It should be. realized that when anhydrides other than acetic anhydride are used for the operation of the present. process, the rate of formation of the corresponding esters may be somewhat different, thus, the time given above for the formation of the acetates will vary. However, using the criteria given in the present description, it is possible to determine with relative ease. the. time. required for the formation of; the other esters. The optimum conditions; for. these other preparatibnsmay be. determinedin a, similarmanner with relative ease.

Other methods are available for observing. the. course of the esterification reaction, for instance, it has been noted; that. the. optical rotation of the reaction mixture gradually changes during the process. Furthermore, there are certain characteristic changes in the microbiological activity: by standard assay methods during. the course of'the reaction. When oxytetracycline is esterified the optical rotation of the mixture gradually increases and whenthe diester formation is practically complete, the rate of increase in the rotation tends toslacken and reacha constant level. There is little tendency to form more highly esterified products than the diacetate at a temperatureof below' about C. during a time period of as much as two or three weeks. If the temperature is raised appreciably above this point different products or mixtures of productsare formed. This is also the casewhen any strongacid' or base is present in the reaction mixture. The" desired monoestors or diesters are. not obtained. Temperatures-lower than 15 C; may be 4 ustlsd for conducting the process, but these have no specific va ue.

The ester derivatives of the tetracycline antibiotics may be isolated from the reaction mixture by removal of the acylating agent and solvent if one is used. Alternatively the product may be precipitated by the addition of a large volume of certain non-solvents for the reaction product, such as diethyl ether or lower aliphatic hydrocarbons. It is often desirable toremove all or part of the excess acetic anhydride and organic solvent if one is used. Care must be exercised during this step to see that the temperature is controlled at about that of the room, that is, not higher than about 35 C. After partial evaporation, which may be conducted under vacuum, the product may be precipitated as indicated above. Complete evaporation yields a solid product directly. The amorphous products, which are generally obtained by precipitation or evaporation, may be crystallized from solvent suchas methanol, ethanol, acetone, or toluene. The crystalline products are bright yellow in color. From the amorphous or crystalline products may be prepared salts with, acids and bases of sutficient strength, preferably mineral acids. or alkali, or alkaline earth metal hydroxides. Examples include the hydrochloride's, sulfates and: phosphates; the sodium, potassium and calcium salts. Generally these may be prepared most simply by addition of the chosen acid or base in an amount sufiicient to neutralize the basic or acidic groups of the antibiotic ester. Solid products may be isolated from aqueous solutions by conventional procedures.

The crystalline diacetate of oxytetracycline has a melting point of about 208 to- 213 C. with decomposition. It is soluble in acetic acid, acetic anhydride, dioxane and acetone, but insoluble in ether or water. Analysis of a sample of the crystalline product gave the following results:

Analysis.Calcd. for CzsHzsOnNz: C, 57.35; H, 5.13; N, 5.15; Acetyl, 15.81. Found: C, 57.59; H, 5.23; N, 5.11;. Acetyl, 15.69.

The optical rotation: is ialzs =2li4 (0.4% in methanol). The infrared absorption curve of the compound shows. peaks. at. 5.70,, 6.03,6.l5, 6.30, 6.35, 2.77, 2.86, 3.10 microns. The ultraviolet absorption spectrum is very similar to that of oxytetracycline under the same conditions.

The. diacctate (and; other diesters) of oxytetracycline may be hydrolyzedto yield in vitro microbiological activity comparable to oxytetracycline by treatment in dilute alkali metal hydroxide solution at room temperature. If one normal sodium hydroxide is used, and samples of the solution are acidified after definite intervals, it is foundthat: 45%. of the microbiological activity (as oxytetracycline) is present after 5 minutes, 62% after lO' minutes, but 12%. after one hour. Apparently some ofthe. antibiotic is. formed and then destroyed during one hour;

When. rabbits. are injected with mg./kg. of the diacetate of oxytetracycline by the intramuscular route the followingaverage blood levels are obtained:

Hours: Meg/ml 0' 0 05 0.66

The monoacetate of oxytetracycline possesses a high order ofi'microbiological activity against a variety of organismsas shownby the. following table, which gives the minimum inhibitory concentration of this compound against: a: group' of microorganisms. under standard con.-

Meg/mg. A. aerogenes (AC 2) 3.25 MT 2 3.25 parent 6 E. coli 6 Proteus 100 Pseudomonas sp. 5 Candida albicans 100 Salmonella typhosa 12 K. pneumoniae 3 Sal. paratyphi A 6 Sal. paratyphi B 6 Str. faecalis 3 Brucella bronchiseptica 3 B. subtillis 0.3 Staph. aureus 3 Ps. aeruginosa Myco. ranae 0.78 smegmatis 0.78 phlei No. 607 1.56 berolinense 3.12 butyricum 3.12

The following examples are given by way of illustration and are not to be considered as the sole embodiments of this invention. It is to be understood that protection hereof is to be limited only by the specific wording of the appended claims.

Example I.-5,12a-diacetyl0xytetracycline Pure, anhydrous, amphoteric oxytetracycline, weighing 10 grams, was mixed with 200 milliliters of pure anhydrous dioxane. This was treated with 800 milliliters of acetic anhydride. The mixture was stored at a temperature of 25 C. for two weeks. Upon concentration of the solution under vacuum at room temperature, crude crystalline oxytetracycline diacetate was obtained. From time to time samples were removed from the solution and the specific rotation was determined. At the beginning of the experiment the optical rotation was +139". After seven days it was +180. At the end of 14 days the rotation had reached +192 and no further change was occurring. Samples of the solution were diluted with acetone and dilute aqueous hydrochloric acid before determining the microbiological assay. It was found that there Was a gradual decrease in the apparent activity of the material to a minimum of about micrograms per milligram of solid at the end of 14 days.

The product was recrystallized from methanol and melted at 208 -213 C. with decomposition.

Example II.-12a-monoacetylchlortetracycline Anhydrous chlortetracycline (2.0 g.) was dissolved in anhydrous dioxan (40 ml.) and the solution was made to 200 ml. with acetic anhydride. The reaction mixture was kept at 25 for 67 hours. It was then evaporated to dryness under reduced pressure. The residue was dissolved in chloroform and a crystalline solid slowly formed. The solid was filtered, washed with toluene and dried in vacuo. It weighed 0.5 g. (bio-assay vs. Klebsiella pneumoniae, 19 'y/mg.). The infrared absorption curve of this product had a peak at 5.8; while the ultraviolet absorption curve in methanol containing hydrogen chloride is almost identical with that of chlortetracycline.

Calculated for C24H25O9N2Cl: 1 acetyl, 8.26; found 8.40.

Example III.1Za-monoacetyloxytetracycline Anhydrous oxytetracycline (20.0 g.) was dissolved in anhydrous dioxan (400 ml.). The solution was made to 2 liters with acetic anhydride, thoroughly mixed and allowed to stand at 25 for hours. The solvent was removed under reduced pressure (35 water bath). The residue was dissolved in an acetone-toluene mixture and evaporated at reduced pressure. A precipitate formed. This was filtered, washed with toluene and dried in vacuo. It Weighed 9 g. (bio-assay vs. Klebsiella pneumoniae 180 /rng) The 12a-rnonoacetyloxytetracycline may be crystallized from methanol. The product has an absorption peak in the infrared region at 5.8 The ultraviolet absorption curve is very similar to that of oxytetracycline.

Calculated for C24H26010N21 1 acetyl, 8.56; found 9.25.

Example lV.-Oxytetracycline propz'onate Two grams of anhydrous oxytetracycline were dissolved in 40 ml. of dioxane and the clear amber solution was made to 200 ml. with propionic anhydride. The solution was allowed to stand at room temperature over a period of 10 days during which the specific rotation changed from +13.5 to +134.2. The reaction mixture was evaporated to dryness under reduced pressure without elevating the temperature. A tan oily residue remained. The residue was dissolved in toluene. The insoluble material was filtered and the filtrate was evaporated to dryness. The product was a vitreous solid. The physical constants of the product showed it to be a propionate of oxytetracycline. It had a microbiological potency against Klebsiella pneumoniae of mcg./rng.

What is claimed is:

1. A process for preparing an esterified tetracycline antibiotic which comprises reacting a tetracycline antibiotic selected from the group consisting of oxytetracycline, chlortetracycline and tetracycline with at least a molar proportion of an alkyl carboxylic acid anhydride having up to 6 carbon atoms in a minor proportion by volume of dioxane at a temperature not exceeding 35 C.

2. A process as claimed in claim 1 wherein the process is conducted at a temperature of from about 15 to about 35 C. for a period not exceeding three weeks.

3. A compound selected from the group consisting of a. 12a-monoester of oxytetracycline, a l2a-monoester of chlortetracycline, a 12a-monoester of tetracycline, and a 5,12a-diester of oxytetracycline, each ester group having up to 6 carbon atoms and being of the formula RCOO- wherein R is alkyl.

4. Oxytetracycline-S,IZa-diacetate.

5. Tetracycline-12a-acetate.

6. Chlortetracycline-lZa-acetate.

7. Oxytetracycline-IZa-acetate.

8. A process as set forth in claim 1 wherein the alkyl carboxylic acid anhydride is acetic anhydride.

References Cited in the file of this patent Hochstein et al.: J. Am. Chem. Soc., vol. 75, November 28, 1953, p. 5468. 

1. A PROCESS FOR PREPARING AN ESTERIFIED TETRACYCLINE ANTIBIOTIC WHICH COMPRISES REATING A TETRACYCLINE ANTI-BIOTIC SELECTED FROM THE GROUP CONSISTING OF OXYTETRACYCLINE, CHLORTETRACYCLINE AND TETRACYCLINE WITH AT LEAST A MOLAR PROPORTION OF AN ALKYL CABOXYLIC ACID ANHYDRIDE HAVING UP TO 6 CARBON ATOMS IN A MINOR PROPORTION BY VOLUME OF DIOXANE AT A TEMPERATURE NOT EXCEEDING 35*C.
 3. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF A 12A-MONOESTER OF OXYTETRACYCLINE, A 12A-MONESTER OF CHLORTETRACYCLINE, A 12-MONESTER OF TETRACYCLINE, AND A 5,12A-DIESTER OF OXYTETRACYCLINE, EACH ESTER GROUP HAVING UP TO 6 CARBON ATOMS AND BEING OF THE FORMULA RCOOWHEREIN R IS ALKYL. 